Vol. 135, No. 1
JOURNAL OF BACTERIOLOGY, July 1978, p. 286-288 0021-9193/78/0135-0286$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Biosynthesis of 8-Aminolevulinic Acid by Blue-Green Algae (Cyanobacteria) J. A. KIPE-NOLT, S. E. STEVENS, JR.,* AND C. L. R. STEVENS Department of Microbiology and Cell Biology, The Pennsylvania State University, University Park,
Pennsylvania 16802 Received for publication 6 March 1978
When levulinic acid, a competitive inhibitor of 6-aminolevulinic acid dehydratase, was added to growing cultures of blue-green algae (cyanobacteria), 6-aminolevulinic acid was excreted into the medium and cell growth was inhibited. 8-Aminolevulinic acid (ALA) is the first identified biosynthetic intermediate that is unique to the tetrapyrrole pathway. ALA is the precursor of heme in animals (17) and bacteria (10) and of chlorophyll in bacteria (5) and plants (7). Troxler and Brown (19) have shown that radioactively labeled ALA is incorporated into the chromophores (phycocyanobilin and phycoerythrobilin) of phycocyanin and phycoerythrin by red algae. Levulinic acid (LA), which is structurally similar to ALA except for the absence of the amino group, has been shown by Nandi and Shemin (15) to be a competitive inhibitor of ALA dehydratase. ALA dehydratase catalyzes the condensation of two molecules of ALA to forn monopyrrole porphobilinogen. Beale (1, 2) demonstrated that LA could be used to induce accumulation of ALA in the medium of a culture of Chlorella. LA has subsequently been used for the same purpose in greening higher plant tissues (3, 4, 8), bacteria (9), Euglena (16), and Cyanidium caldarium (11). The present study reports the use of LA to demonstrate the synthesis of ALA in blue-green algae (cyanobacteria). We believe this to be the first direct demonstration of ALA biosynthesis by the bluegreen algae. Angmenellum quadruplicatum PR-6, Anacystis marina 6, and Coccochloris elabens Di (20) were grown in medium A (18) at 39°C. Anacystis nidulans TX-20 (13) was grown in medium C (18) at 390C. Nostoc muscorum, Nostoc sp. strain MAC, and Anabaena flos-aquae were grown in medium B (18) at 350C. Continuous agitation and C02 were provided by bubbling 4% (vol/vol) C02 in air through all cultures. Illumination consisted of four (two on each side of the bath) F24T12 CW/HO fluorescent lamps. Growth was measured turbidimetrically with a Bausch and Lomb Spectronic 20 spectrometer at 550 nm or by viable cell count (21). A stock solution of 1 M LA was prepared, adjusted to pH 7.6 with NaOH, filter sterilized
(0.45-um Millipore filter), and added to growing algal cultures. ALA was condensed with acetylacetone and then estimated as the 2-methyl3-acetyl-4-(3-propionic acid) pyrrole derivative with Ehrlich reagent (14). ALA produced byA. quadruplicatum PR-6 in the presence of 60 mM LA was verified against genuine ALA on silica gel thin-layer plates. ALA was isolated from spent medium (6, 11), converted to the ALA pyrrole (14), developed on thin-layer plates with n-butanol-1 N NH40H (1:1, vol/vol), and visualized with Ehrlich reagent. The time course of ALA production by strain PR-6 in the presence of 60 mM LA is shown in Fig. 1. Growth or increase in cell number was completely inhibited when this concentration of LA was added to a culture with an initial cell number of 3.0 x 107 cells per ml. Prior to addition of LA, extracellular ALA was not detectable in the culture fluid. However, upon addition of LA, growth ceased and ALA began to accumulate. Continuous accumulation was observed for up to 48 h, suggesting that ALA does not feedback inhibit its own synthesis. The amounts of ALA excreted into the medium by strain PR-6 in 20 h when different concentrations of LA were added to different initial cell densities are shown in Fig. 2. Five different concentrations of LA were added to five different culture tubes, all with the same initial cell concentration. After 20 h, the amount of ALA that had accumulated in the medium in each tube was determined. This experiment was repeated four times using four different initial cell concentrations. Cell growth and the amount of ALA accumulated depended on LA concentration and initial cell number. The saturation-type kinetics of ALA production at different LA concentrations observed in this study were similar to those observed in maize leaves (12). However, they were unlike that observed in a number of other studies in-
286
NOTES
VOL. 135, 1978
287
cluding bean leaves (12), barley leaves (4), cucumber cotyledons (4), and C. caldarium (11); in this last case there was an LA concentration at which maximum ALA was produced, and at higher LA concentrations less ALA was produced. In cultures with LA concentrations above satA/' uration, cell growth (division) was completely 'L inhibited. However, if the cells were centrifuged and fresh medium without LA was added, the cells recovered after a short lag and growth was 0 normal. In cultures with LA concentrations be- a low saturation, there was an increase in growth rate with a decrease in LA concentration. o c The compound that was excreted by strain PR-6 in the presence of LA formed an Ehrlichpositive pyrrole when boiled in acetylacetone. The pyrrole had an Rf of 0.2 in butanol-ammonia, which corresponded to standard ALA-pyrso 0 20 40 6 1o Levulinic Acid Concentration (mM) role. This verified that the compound excreted by strain PR-6 in the presence of LA was ALA. FIG. 2. Nanomoles of ALA produced per milliliter The usefulness of LA for inhibiting ALA de- in 20 h when different concentrations of LA were hydratase was extended to Nostoc sp. strain added to different initial cell densities. Initial cell MAC, A. nidulans TX-20, C. elabens Di, A. numbers: (0) 1.4 x 167 cells per ml; (0) 2.7 x 107 cells marina 6, A. flos-aquae, and N. muscorum. per ml; (U) 5.7 x 167 cells per ml; (A) 8.1 x 167 cells These include unicellular and filamentous algae, per ml. marine and freshwater algae, nitrogen-fixing and non-nitrogen-fixing algae, obligate autotrophs, and a facultative heterotroph. Thus, the accumulation of ALA in the presence of LA seems to a general characteristic among blue-green 20n S0 be algae. Gratifyingly unsurprising, our results remove a tacit assumption and exchange it for a fact.
4/
30
15
This work was supported by Public Health Service grant GM-23524 from the National Institute of General Medical Sciences.
LITERATURE CITED 40
2
4~~~~~~~~~
0
Time (hr)
FIG. 1. Production ofALA and growth of a culture of strain PR-6 in the presence of 60 mM LA. (A) Nanomoles of ALA per milliliter; (0) cell number of LA-treated culture; (U) cell number of culture without LA.
1. Beale, S. I. 1970. The biosynthesis of 8-aminolevulinic acid in Chlorella. Plant Physiol. 45:505-506. 2. Beale, S. I. 1971. Studies on the biosynthesis and metabolism of 8-aminolevulinic acid in Chlorella. Plant Physiol. 48:316-319. 3. Beale, S. I., and P. A. Castelfranco. 1973. '4C incorporation from exogenous compounds into 8-aminolevulinic acid by greening cucumber cotyledons. Biochem. Biophys. Res. Commun. 52:143-149. 4. Beale, S. I., and P. A. Castelfranco. 1974. The biosynthesis of 8-aminolevulinic acid in higher plants. I. Accumulation of 8-aminolevulinic acid in greening plant
tissues. Plant Physiol. 53:291-296. 5. Brunham, B. F., and J. Lascelles. 1963. Control of porphyrin biosynthesis through a negative-feedback mechanism. Studies with preparations of 8-aminolevulate synthetase and B-ALA dehydratase from Rhodopseudomonas spheroides. Biochem. J. 87:462-472. 6. Duggan, J., and M. Gassman. 1974. Induction of porphyrin synthesis in etiolated bean leaves by chelators of iron. Plant Physiol. 53:206-215. 7. Granick, S. 1961. Magnesium protoporphyrin monoester and protoporphyrin monomethyl ester in chlorophyll biosynthesis. J. Biol. Chem. 236:1168-1172. 8. Harel, E., and S. Klein. 1972. Light dependent formation of 6-aminolevulinic acid in etiolated leaves of higher
288
NOTES
plants. Biochem. Biophys. Res. Commun. 49:364-370. 9. Ho, Y. K., and J. Lascelles. 1971. 6-aminolevulinic acid dehydratase of Spirillum itersonii and the regulation of tetrapyrrole synthesis. Arch. Biochem. Biophys. 144:734-740. 10. Jacobs, N. J. 1977. Biosynthesis of heme, p. 125-148. In J. B. Neilands (ed.), Microbial iron metabolism. Academic Press Inc., New York. 11. Jurgenson, J. E., S. L. Beale, and R. F. Troxler. 1976. Biosynthesis of 8-aminolevulinic acid in the unicellular Rhodophyte, Cyanidium caldarium. Biochem. Biophys. Res. Commun. 69:149-157. 12. Klein, S., E. Harel, E. Ne'eman, E. Katz, and E. Meller. 1975. Accumulation of 8-aminolevulinic acid and its relation to chlorophyll synthesis and development of plastid structure in greening leaves. Plant Physiol. 56:486-496. 13. Kratz, W. A., and J. Myers. 1955. Nutrition and growth of several blue-green algae. Am. J. Bot. 42:282-287. 14. Mauzerall, D., and S. Granick. 1956. The occurrence and determination of 6-aminolevulinic acid and porphobilinogen in urine. J. Biol. Chem. 219:435-446.
J. BACTERIOL. 15. Nandi, D. L., and D. Shemin. 1968. 8-Aminolevulinic acid dehydratase of Rhodopseudomonas spheroides. III. Mechanism of porphobilinogen synthesis. J. Biol. Chem. 243:1236-1242. 16. Richard, F., and V. Nigon. 1972. La production d'acide 8-aminolevulinique au cours du verdissement d'euglenes etiolees et ses relations avec la photosynthese. C. R. Acad. Sci. Paris Ser. D 274:1307-1310. 17. Shemin, D., and C. S. Russell. 1953. O-Aminolevulinic acid, its role in the biosynthesis of porphyrins and purines. J. Am. Chem. Soc. 75:4873-4874. 18. Stevens, S. E., Jr., C. 0. P. Patterson, and J. Myers. 1973. The production of hydrogen peroxide by bluegreen algae: a survey. J. Phycol. 9:427-430. 19. Troxler, R. F., and A. S. Brown. 1975. Metabolism of 8-aminolevulinic acid in red and blue-green algae. Plant Physiol. 55:463-467. 20. Van Baalen, C. 1962. Studies on marine blue-green algae. Bot. Mar. 4:129-139. 21. Van Baalen, C. 1967. Further observations on growth of single cells of coccoid blue-green algae. J. Phycol. 3:154-157.
JOURNAL OF BACTERIOLOGY, July 1978, p. 286-288 0021-9193/78/0135-0286$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Biosynthesis of 8-Aminolevulinic Acid by Blue-Green Algae (Cyanobacteria) J. A. KIPE-NOLT, S. E. STEVENS, JR.,* AND C. L. R. STEVENS Department of Microbiology and Cell Biology, The Pennsylvania State University, University Park,
Pennsylvania 16802 Received for publication 6 March 1978
When levulinic acid, a competitive inhibitor of 6-aminolevulinic acid dehydratase, was added to growing cultures of blue-green algae (cyanobacteria), 6-aminolevulinic acid was excreted into the medium and cell growth was inhibited. 8-Aminolevulinic acid (ALA) is the first identified biosynthetic intermediate that is unique to the tetrapyrrole pathway. ALA is the precursor of heme in animals (17) and bacteria (10) and of chlorophyll in bacteria (5) and plants (7). Troxler and Brown (19) have shown that radioactively labeled ALA is incorporated into the chromophores (phycocyanobilin and phycoerythrobilin) of phycocyanin and phycoerythrin by red algae. Levulinic acid (LA), which is structurally similar to ALA except for the absence of the amino group, has been shown by Nandi and Shemin (15) to be a competitive inhibitor of ALA dehydratase. ALA dehydratase catalyzes the condensation of two molecules of ALA to forn monopyrrole porphobilinogen. Beale (1, 2) demonstrated that LA could be used to induce accumulation of ALA in the medium of a culture of Chlorella. LA has subsequently been used for the same purpose in greening higher plant tissues (3, 4, 8), bacteria (9), Euglena (16), and Cyanidium caldarium (11). The present study reports the use of LA to demonstrate the synthesis of ALA in blue-green algae (cyanobacteria). We believe this to be the first direct demonstration of ALA biosynthesis by the bluegreen algae. Angmenellum quadruplicatum PR-6, Anacystis marina 6, and Coccochloris elabens Di (20) were grown in medium A (18) at 39°C. Anacystis nidulans TX-20 (13) was grown in medium C (18) at 390C. Nostoc muscorum, Nostoc sp. strain MAC, and Anabaena flos-aquae were grown in medium B (18) at 350C. Continuous agitation and C02 were provided by bubbling 4% (vol/vol) C02 in air through all cultures. Illumination consisted of four (two on each side of the bath) F24T12 CW/HO fluorescent lamps. Growth was measured turbidimetrically with a Bausch and Lomb Spectronic 20 spectrometer at 550 nm or by viable cell count (21). A stock solution of 1 M LA was prepared, adjusted to pH 7.6 with NaOH, filter sterilized
(0.45-um Millipore filter), and added to growing algal cultures. ALA was condensed with acetylacetone and then estimated as the 2-methyl3-acetyl-4-(3-propionic acid) pyrrole derivative with Ehrlich reagent (14). ALA produced byA. quadruplicatum PR-6 in the presence of 60 mM LA was verified against genuine ALA on silica gel thin-layer plates. ALA was isolated from spent medium (6, 11), converted to the ALA pyrrole (14), developed on thin-layer plates with n-butanol-1 N NH40H (1:1, vol/vol), and visualized with Ehrlich reagent. The time course of ALA production by strain PR-6 in the presence of 60 mM LA is shown in Fig. 1. Growth or increase in cell number was completely inhibited when this concentration of LA was added to a culture with an initial cell number of 3.0 x 107 cells per ml. Prior to addition of LA, extracellular ALA was not detectable in the culture fluid. However, upon addition of LA, growth ceased and ALA began to accumulate. Continuous accumulation was observed for up to 48 h, suggesting that ALA does not feedback inhibit its own synthesis. The amounts of ALA excreted into the medium by strain PR-6 in 20 h when different concentrations of LA were added to different initial cell densities are shown in Fig. 2. Five different concentrations of LA were added to five different culture tubes, all with the same initial cell concentration. After 20 h, the amount of ALA that had accumulated in the medium in each tube was determined. This experiment was repeated four times using four different initial cell concentrations. Cell growth and the amount of ALA accumulated depended on LA concentration and initial cell number. The saturation-type kinetics of ALA production at different LA concentrations observed in this study were similar to those observed in maize leaves (12). However, they were unlike that observed in a number of other studies in-
286
NOTES
VOL. 135, 1978
287
cluding bean leaves (12), barley leaves (4), cucumber cotyledons (4), and C. caldarium (11); in this last case there was an LA concentration at which maximum ALA was produced, and at higher LA concentrations less ALA was produced. In cultures with LA concentrations above satA/' uration, cell growth (division) was completely 'L inhibited. However, if the cells were centrifuged and fresh medium without LA was added, the cells recovered after a short lag and growth was 0 normal. In cultures with LA concentrations be- a low saturation, there was an increase in growth rate with a decrease in LA concentration. o c The compound that was excreted by strain PR-6 in the presence of LA formed an Ehrlichpositive pyrrole when boiled in acetylacetone. The pyrrole had an Rf of 0.2 in butanol-ammonia, which corresponded to standard ALA-pyrso 0 20 40 6 1o Levulinic Acid Concentration (mM) role. This verified that the compound excreted by strain PR-6 in the presence of LA was ALA. FIG. 2. Nanomoles of ALA produced per milliliter The usefulness of LA for inhibiting ALA de- in 20 h when different concentrations of LA were hydratase was extended to Nostoc sp. strain added to different initial cell densities. Initial cell MAC, A. nidulans TX-20, C. elabens Di, A. numbers: (0) 1.4 x 167 cells per ml; (0) 2.7 x 107 cells marina 6, A. flos-aquae, and N. muscorum. per ml; (U) 5.7 x 167 cells per ml; (A) 8.1 x 167 cells These include unicellular and filamentous algae, per ml. marine and freshwater algae, nitrogen-fixing and non-nitrogen-fixing algae, obligate autotrophs, and a facultative heterotroph. Thus, the accumulation of ALA in the presence of LA seems to a general characteristic among blue-green 20n S0 be algae. Gratifyingly unsurprising, our results remove a tacit assumption and exchange it for a fact.
4/
30
15
This work was supported by Public Health Service grant GM-23524 from the National Institute of General Medical Sciences.
LITERATURE CITED 40
2
4~~~~~~~~~
0
Time (hr)
FIG. 1. Production ofALA and growth of a culture of strain PR-6 in the presence of 60 mM LA. (A) Nanomoles of ALA per milliliter; (0) cell number of LA-treated culture; (U) cell number of culture without LA.
1. Beale, S. I. 1970. The biosynthesis of 8-aminolevulinic acid in Chlorella. Plant Physiol. 45:505-506. 2. Beale, S. I. 1971. Studies on the biosynthesis and metabolism of 8-aminolevulinic acid in Chlorella. Plant Physiol. 48:316-319. 3. Beale, S. I., and P. A. Castelfranco. 1973. '4C incorporation from exogenous compounds into 8-aminolevulinic acid by greening cucumber cotyledons. Biochem. Biophys. Res. Commun. 52:143-149. 4. Beale, S. I., and P. A. Castelfranco. 1974. The biosynthesis of 8-aminolevulinic acid in higher plants. I. Accumulation of 8-aminolevulinic acid in greening plant
tissues. Plant Physiol. 53:291-296. 5. Brunham, B. F., and J. Lascelles. 1963. Control of porphyrin biosynthesis through a negative-feedback mechanism. Studies with preparations of 8-aminolevulate synthetase and B-ALA dehydratase from Rhodopseudomonas spheroides. Biochem. J. 87:462-472. 6. Duggan, J., and M. Gassman. 1974. Induction of porphyrin synthesis in etiolated bean leaves by chelators of iron. Plant Physiol. 53:206-215. 7. Granick, S. 1961. Magnesium protoporphyrin monoester and protoporphyrin monomethyl ester in chlorophyll biosynthesis. J. Biol. Chem. 236:1168-1172. 8. Harel, E., and S. Klein. 1972. Light dependent formation of 6-aminolevulinic acid in etiolated leaves of higher
288
NOTES
plants. Biochem. Biophys. Res. Commun. 49:364-370. 9. Ho, Y. K., and J. Lascelles. 1971. 6-aminolevulinic acid dehydratase of Spirillum itersonii and the regulation of tetrapyrrole synthesis. Arch. Biochem. Biophys. 144:734-740. 10. Jacobs, N. J. 1977. Biosynthesis of heme, p. 125-148. In J. B. Neilands (ed.), Microbial iron metabolism. Academic Press Inc., New York. 11. Jurgenson, J. E., S. L. Beale, and R. F. Troxler. 1976. Biosynthesis of 8-aminolevulinic acid in the unicellular Rhodophyte, Cyanidium caldarium. Biochem. Biophys. Res. Commun. 69:149-157. 12. Klein, S., E. Harel, E. Ne'eman, E. Katz, and E. Meller. 1975. Accumulation of 8-aminolevulinic acid and its relation to chlorophyll synthesis and development of plastid structure in greening leaves. Plant Physiol. 56:486-496. 13. Kratz, W. A., and J. Myers. 1955. Nutrition and growth of several blue-green algae. Am. J. Bot. 42:282-287. 14. Mauzerall, D., and S. Granick. 1956. The occurrence and determination of 6-aminolevulinic acid and porphobilinogen in urine. J. Biol. Chem. 219:435-446.
J. BACTERIOL. 15. Nandi, D. L., and D. Shemin. 1968. 8-Aminolevulinic acid dehydratase of Rhodopseudomonas spheroides. III. Mechanism of porphobilinogen synthesis. J. Biol. Chem. 243:1236-1242. 16. Richard, F., and V. Nigon. 1972. La production d'acide 8-aminolevulinique au cours du verdissement d'euglenes etiolees et ses relations avec la photosynthese. C. R. Acad. Sci. Paris Ser. D 274:1307-1310. 17. Shemin, D., and C. S. Russell. 1953. O-Aminolevulinic acid, its role in the biosynthesis of porphyrins and purines. J. Am. Chem. Soc. 75:4873-4874. 18. Stevens, S. E., Jr., C. 0. P. Patterson, and J. Myers. 1973. The production of hydrogen peroxide by bluegreen algae: a survey. J. Phycol. 9:427-430. 19. Troxler, R. F., and A. S. Brown. 1975. Metabolism of 8-aminolevulinic acid in red and blue-green algae. Plant Physiol. 55:463-467. 20. Van Baalen, C. 1962. Studies on marine blue-green algae. Bot. Mar. 4:129-139. 21. Van Baalen, C. 1967. Further observations on growth of single cells of coccoid blue-green algae. J. Phycol. 3:154-157.
